The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Commemorative Booklet for CAAT 20th Anniversary Symposium

Rhythmically Stretching Dynamic Cell Culture: Alternative Approach to Study Particle-Induced Cell Injury

Akira Tsuda
Harvard School of Public Health, Boston

Exposure to airborne particulates is strongly associated with lung injury, but the molecular and cellular mechanisms for this association are not fully understood. The ultimate goal of this project is to improve our understanding of particle-induced pathogenesis in the lung. Specifically, we are investigating the effect of the physical insults exerted by particles on alveolar epithelial cells. Our central hypothesis is that the mechanical contact between particles and the epithelial cells plays an important role in lung injury, and that the physical stimuli exerted on cells by particles may be greatly enhanced by cyclic motion; these combined effects may trigger subsequent cell responses.

In Study 1, monolayers of the human alveolar epithelial cell line, A549, were rhythmically stretched with physiologically relevant tidal breathing conditions (strain of 5% at 10 X/ min) with glass or crocidolite asbestos fibers. Cyclic stretching significantly increased interleukin-8 (IL-8) production in the fiber-treated cultures, suggesting that the physical stress of the cells caused by the fibers was indeed enhanced by the motion. Coating of the asbestos fibers with fibronectin (FN) further increased the fiber-induced cell response when the cells were cyclically stretched. This response was reduced significantly, however, by introducing soluble RGD peptides into the culture medium prior to fiber treatment, which specifically blocks fibronectin binding to integrin receptors. These results suggest that adhesive interactions between protein-coated fibers and cell surface molecules are involved in the fiber-induced pathogenic process.

Study 2 examined changes in "global" gene expression profiles during fiber treatments using microarray analysis. Twenty genes were consistently upregulated (> 2 fold) and 24 genes were downregulated in stretched cells with particles in two replicate trials. Genes upregulated included IL-8, stress genes (antioxidant, heat shock proteins), and genes involved with translational machinery. The majority of downregulated genes were for cytoskeletal proteins. Array screening has identified a number of gene products modulated in A549 cells by cell stretching and contact with FNcoated particles.

Studies were done with collaboration with Drs. Stringer, Mijailovich (Study-1), Al-Mutairi, and Paulauskis (Study-2).